DESCRIPTION: This project uses methods and logic of molecular biology and genetics to investigate two components of the translation apparatus that are responsible for the rules of the genetic code. These components--transfer RNAs (tRNAs) and aminoacyl tRNA synthestases-- appeared early in evolution and became essential for all life forms. The code is established in aminoacylation reactions, whereby each of the twenty amino acids is attached to its cognate tRNA that bears the anticodon triplet of the code for that amino acid. There typically is one distinct synthetase for each amino acid. In the proposed work, much effort is directed at using genetic approached to study how the fine structure recognition of amino acids is achieved by the tRNA synthetases. This high level of discrimination is essential for establishing and maintaining the accuracy of the code. For some of the enzymes, the cognate tRNA plays a key role in achieving this highly differentiated fine structure discrimination of closely similar amino acids. The role of the tRNA is to direct a misactivated amino acid from the active site to a second, editing site, where a potential error of aminoacylation is eliminated. Genetic approaches are proposed to explore the consequences in vivo of mutations in the center for editing, and to demonstrate the misincorporation of amino acids into proteins as they are synthesized in the cell. In further work, semi-artificial tRNA synthetases will be created and studied in vivo. This work is intended to provide insight into how specific systems of aminoacylation can be assembled from simple pieces. Finally, recent work has established other roles for tRNA synthetases in higher organisms, particularly in human cells. These roles are to be investigated further using genetic screens. Because tRNA synthetases are essential proteins, they are being pursued as targets for new classes of antibiotics. Moreover, the recent demonstration of novel roles for these proteins in cellular signaling mechanisms suggests that other heath-related applications will emerge as more basic information is obtained on these systems.